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Diss Factsheets

Administrative data

Description of key information

Skin sensitisation in vivo: OECD 406, GPMT, 1998: negative for skin sensitisation

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 Oct - 03 Nov 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
Adopted in 1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
Adopted in 1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPP 81-6 (Skin Sensitisation)
Version / remarks:
Adopted in 1984
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japan/M.A.F.F. guideline, 59 NohSan No. 4200
Version / remarks:
Adopted in 1985
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
A non-LLNA study is available that was performed prior to the current data requirements, stipulated in Regulation (EC) No 1907/2006, and before the LLNA test method was available (the LLNA was adopted in 2002). In accordance with Regulation (EC) No 1907/2006, the data was included to avoid unnecessary testing.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France, Saint-Aubin-lès-Elbeuf, France
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 3 months
- Body weight at study initiation: males: 349 ± 10 g, females: 356 ± 18 g
- Housing: individually, in polycarbonate cages (48 cm x 27 cm x 20 cm) with dust-free sawdust (SICSA, Alfortville, France)
- Diet: 106 pelleted diet (U.A.R., Villemoisson-sur-Orge, France), ad libitum
- Water: filtered water of drinking water quality, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 30 - 70
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

- IN-LIFE DATES: From: 10 Oct 1997 To: 03 Nov 1997
Route:
intradermal and epicutaneous
Vehicle:
paraffin oil
Concentration / amount:
intradermal injection: 1% (w/w); topical application: undiluted test substance
Day(s)/duration:
On Day 1, animals received 3 pairs of intradermal injections. 6 days thereafter (Day 7), the injection sites were pre-treated with 10% SDS. 24 h later (Day 8), the vehicle or the test substance were placed on the pre-treated skin and fixed for 48 h.
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
500 mg of the undiluted test substance were applied to the posterior right flank
Day(s)/duration:
1 day
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
control group: 5 males, 5 females
treatment group: 10 males, 10 females
Details on study design:
RANGE FINDING TEST
A preliminary test was conducted in one male and one female in order to determine the concentrations to be tested in the main study.

By intradermal route:
- 24 h before treatment, the dorsal region of the animals was clipped.
- Intradermal administrations of the test substance formulations (0.1 mL) at 1 and 0.1% (w/w) in paraffin oil with or without FCA were performed in the interscapular region.
- Cutaneous reactions were evaluated approximately 24, 48 h and 6 days after the injections.

By cutaneous route:
- 24 h before treatment, both flank regions of the animals were clipped.
- 500 mg of the undiluted test substance or 0.5 mL of test substance formulations at 20% (w/w) in paraffin oil were placed on a moistened (undiluted test substance) or on a dry gauze pad (approximately 4 cm²), which was then applied to the skin and held in place by an occlusive dressing for 24 h.
- Cutaneous reactions were evaluated approximately 24 and 48 h after removal of the dressings.

Criteria for selection of concentrations - the following criteria were used:
- The concentrations should be well-tolerated systemically and locally.
- Intradermal injections should cause moderate irritant effect (no necrosis or ulceration of the skin).
- Cutaneous application for the induction should cause at most weak or moderate skin reactions or be the maximal practicable concentration.
- Cutaneous application for the challenge phase should be the highest concentration which does not cause irritant effects.

Results:
Administration by intradermal route (24 h, 48 h and 6 days):
The test substance (with or without FCA) caused an irritant effect in the male and irritant to slightly irritant effect in the female at a concentration of 1% (w/w). At a concentration of 0.1% (w/w), the test substance induced an irritant effect in the male, but only a slightly irritant effect in the female. Co-administration of 0.1% (v/v) FCA only caused a slightly irritant effect in the female and irritant effect in the male. Based on this data and in order to respect the criteria for the selection of concentrations, the concentration chosen for the main study was 1% (w/w).

Administration by cutaneous route (24 h and 48 h):
At a concentration of 100% (undiluted) or 20% (w/w), the test substance did not cause erythema or oedema at the application sites. In order to respect the criteria for the selection of concentrations, the concentration chosen for the topical application of the induction phase (Day 8) and for the challenge application was 100% (undiluted).

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal and epicutaneous, respectively)
- Exposure period: single injection (intradermal) and 48 h (epicutaneous)

- Test groups:
Intradermal (3 pairs of injections):
Injection 1: 1:1 mixture (v/v) FCA/0.9% NaCl
Injection 2: test substance in 1% (w/w) paraffin oil
Injection 3: test substance in a 1:1 mixture (v/v) FCA/0.9% NaCl
Epicutaneous: undiluted test substance (100%)

- Control group:
Intradermal (3 pairs of injections):
Injection 1: 1:1 mixture (v/v) FCA in 0.9% NaCl solution
Injection 2: paraffin oil
Injection 3: paraffin oil in a 1:1 mixture (v/v) FCA/0.9% NaCl
Epicutaneous: paraffin oil

- Site: shoulder region (intradermal and epicutaneous)
- Frequency of applications: twice (Day 1 and Day 8)
- Duration: Days 0 - 8
- Concentrations: intradermal 1%, epicutaneous 100%

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day of challenge: 22
- Exposure period: 24 h
- Test groups: test substance and paraffin oil only
- Control group: test substance and paraffin oil only
- Site: posterior right flank (test substance) and posterior left flank (vehicle)
- Concentrations: 100%
- Evaluation (h after challenge): 24 and 48 h

OTHER:
On Day 7 (24 h before epicutaneous application of the test substance), the interscapular area of the animals was clipped and they were treated with 0.5 mL of sodium lauryl sulphate (10%, w/w) in vaseline in order to induce local irritation.
Challenge controls:
The control group is actually a challenge control.
Positive control substance(s):
yes
Remarks:
2,4-dinitrochlorobenzene (0.1 and 1% (w/w)) and mercaptobenzothiazole (1 and 20% (w/w))
Positive control results:
The test substance 2,4-dinitrochlorobenzene at the concentration of 1% (w/w) induced positive skin sensitisation reactions in 9/10 animals (90%), thus meeting the reliability criteria for the GPMT (≥ 30% positive response).
The test substance mercaptobenzothiazole at the concentration of 20% (w/w) induced positive skin sensitisation reactions in 3/10 animals (30%), thus meeting the reliability criteria for the GPMT (≥ 30% positive response).
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
induction: 0%; challenge: 100%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
induction: 1 and 100%; challenge: 100%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
2,4-dinitrochlorobenzene: induction: 0.1 and 1%; challenge: 1%
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
10/10: well-defined erythema
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
mercaptobenzothiazole: induction: 1 and 20%; challenge: 20%
No. with + reactions:
5
Total no. in group:
10
Clinical observations:
3/10: very slight erythema; 2/10: well-defined erythema
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
induction: 0%; challenge: 100%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
induction: 1 and 100%; challenge: 100%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
2,4-dinitrochlorobenzene: induction: 0.1 and 1%; challenge: 1%
No. with + reactions:
9
Total no. in group:
10
Clinical observations:
1/10: slight erythema; 4/10: well-defined erythema; 1/10: moderate to severe erythema; 1/10: slight edema; 4/10: scoring was masked by dryness of skin
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
mercaptobenzothiazole: induction: 1 and 20%; challenge: 20%
No. with + reactions:
3
Total no. in group:
10
Clinical observations:
2/10: very slight erythema; 1/10: well-defined erythema; 1/10: moderate to severe erythema; 1/10: slight edema
Remarks on result:
positive indication of skin sensitisation

No clinical signs and no mortalities were observed during the study.

The body weight gain of the treated animals was normal when compared to the control animals.

On Day 10, after the cutaneous application of the induction period, signs of irritation (erythema) were observed at the interscapular test site in the control and treated groups.

Following challenge exposure no cutaneous reactions were observed.

Interpretation of results:
GHS criteria not met
Conclusions:
CLP: not classified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The skin sensitising potential of the test substancewas investigated in guinea pigs in accordance with OECD test guideline (TG) 406 and GLP (M-192475-01-1, 1998). The induction and challenge treatments were carried out according to the maximisation method as described in OECD TG 406.

In a preliminary test, suitable treatment concentrations for the main study were determined in two animals. For the intradermal route, 0.1 mL of test substance formulations in paraffin oil at concentrations of 1 and 0.1% (w/w) were used and administered with or without Freund’s Complete Adjuvant (FCA) to the clipped skin of the interscapular region of 1 male and 1 female. Cutaneous reactions were evaluated approximately 24, 48 h and 6 days after the injections. Based on the induction of moderate irritant effects on the skin but no necrosis or ulceration, a test substance concentration of 1% in paraffin oil (w/w) was chosen for intradermal injections in the main study. For the cutaneous route, 500 mg of the undiluted test substance or 0.5 mL of the test substance at 20% (w/w) in paraffin oil were applied to the clipped skin of the interscapular region of the animals for 24 h using an occlusive dressing. Cutaneous reactions were evaluated approximately 24 and 48 h after removal of the dressings. Since the mean erythema and edema scores of the tested animals over 24 and 48 h post-application were both 0 and no primary dermal irritation was observed on the skin of the treated animals up to a test substance concentration of 100%, this concentration was selected for both the topical application of the induction phase and for the challenge application in the main study.

In the induction phase of the main study, intradermal injections of the test substance at 1% (w/w) and/or FCA were applied into the clipped interscapular area of 10 males and 10 females. A control group, consisting of 5 males and 5 females, was injected with vehicle only and/or FCA. After 7 days and before epicutaneous application of the test substance, all animals were treated with 0.5 mL of sodium lauryl sulphate (10%, w/w) in vaseline in order to induce local irritation. On Day 8, a 48-h epicutaneous induction treatment with the undiluted test substance or vehicle only was performed in the treated or control animals on the regions of intradermal injections. One hour after removal of the occlusive dressing, signs of irritation (erythema) were observed at the interscapular test site in animals of the control and treated group. On Day 22, the challenge treatment was performed by topical application of the undiluted test substance and the vehicle to all animals for 24 h. Skin reactions were evaluated 24 and 48 h after the challenge application. During the study no test substance-related clinical signs and no effects on body weight gain were observed. No cutaneous reactions were provoked by the challenge treatment with the undiluted test substance in none of the animals of the test and control groups. Therefore, the test substance had no sensitising effect on guinea pigs under the chosen experimental conditions.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The available data on skin sensitisation of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.